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Surface modification by poly(ethylene glycol) (PEG) onto gene carrier prepared through the electrostatic assembly of pDNA and polycation (polyplex) is a widely acknowledged strategy to advance their systemic application. In this regard, PEG crowdedness on the polyplex surface should give important contribution in determining blood circulation property; however its accurate quantification has never been demonstrated. We report here the first successful determination of PEG crowdedness for PEGylated polyplexes (polyplex micelle) formed from PEG–poly(l-lysine) block copolymers (PEG–PLys) and plasmid DNA (pDNA). Tethered PEG chains were found to adopt mushroom and even squeezed conformation by modulating PEG crowdedness through PLys segment length. Energetic analysis was conducted on the polyplex micelle to elucidate effect of PEG crowdedness on shape and clarify its essential role in regulating packaging structure of pDNA within the polyplex micelle. Furthermore, the PEG crowdedness significantly correlated ...

The cellular internalization of defined PMMA nanoparticles was investigated. For this purpose, the biocompatible copolymer p(MMA-stat-MAA)0.91:0.09 was synthesized by RAFT polymerization and labeled with three different fluorescent dyes (λEx = 493, 557, and 653 nm). Nanoparticles were formulated from the differently labeled copolymers into samples with relatively narrow size distribution (diameter d 300 nm) under appropriate conditions of nanoprecipitation and were subsequently characterized by DLS and SEM. Mixtures of the differently sized nanoparticle samples were applied for internalization studies using monolayer cultured HeLa cells. The localization of the nanoparticles was detected after certain time points up to 24 h by CLSM, using LysoTracker as a marker for late endosomes and lysosomes. In investigations by flow cytometry, a fast uptake of medium sized nanoparticles was found, whereas the large and small nanoparticles exhibited a slower internalization. However, small and medium sized nanoparticles were detected in the late endosomes/lysosomes, whereas the large nanoparticles exhibit little co-localization with LysoTracker. Moreover, it could be shown by using different inhibitors for clathrin-dependent (chlorpromazine), caveolin-dependent (filipin III) endocytosis and macropinocytosis (EIPA) that nanoparticles with d 300 nm were internalized via macropinocytosis.

For systemic siRNA delivery into tumor tissues, a safe and efficient vehicle is strongly required. Therefore, we designed a block copolymer of detachable poly(ethylene glycol) (PEG) polycation bearing low pKa amines and hydrophobic moieties in the side chain to develop a smart siRNA complex possessing biocompatibility, high complex stability, and endosomal escaping functionality. A disulfide linkage (-SS-) was inserted as a linker between PEG and a cationic polyaspartamide derivative, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), with a flanking stearoyl moiety, where PAsp(DET) segment provides the excellent ability of endosome destabilization by direct interaction with the membrane. The resulting polymer, stearoyl PEG-SS-PAsp(DET), was confirmed to form the siRNA complex with an environment-responsive PEG palisade, which was detached from the complex surface under reductive conditions mimicking tumor tissues and cytoplasm because of the disulfide cleavage. The smart siRNA complex allowed significant gene silencing against cultured pancreatic cancer cells without considerable cytotoxicity and erythrocyte disruption, whereas such significant gene silencing was not observed in a control siRNA complex without the disulfide linkage. This enhanced gene silencing activity might be because of the enhanced cellular uptake and subsequent translocation of siRNA into cytoplasm facilitated by PEG detachment around and/or in the cancer cells. Further, intravital real-time confocal laser scanning microscopic observation revealed the effect of hydrophobic stearoyl modification on the stabilization of the siRNA complex for longevity in the blood. Significant in vivo gene silencing of the smart siRNA complex was achieved by systemic administration of vascular endothelial growth factor siRNA in a mouse model bearing a subcutaneous pancreatic tumor, leading to 40% regression in tumor growth. These results demonstrate the strong potential of stearoyl PEG-SS-PAsp(DET) as a vehicle for systemic delivery of siRNA in cancer therapy.

Surface modification using poly(ethylene glycol) (PEG) is a widely used strategy to improve the biocompatibility of cationic polymer-based nonviral gene vectors (polyplexes). A novel method based on intravital real-time confocal laser scanning microscopy (IVRTCLSM) was applied to quantify the dynamic states of polyplexes in the bloodstream, thereby demonstrating the efficacy of PEGylation to prevent their agglomeration. Blood flow in the earlobe blood vessels of experimental animals was monitored in a noninvasive manner to directly observe polyplexes in the circulation. Polyplexes formed distinct aggregates immediately after intravenous injection, followed by interaction with platelets. To quantify aggregate formation and platelet interaction, the coefficient of variation and Pearson's correlation coefficient were adopted. In contrast, polyplex micelles prepared through self-assembly of plasmid DNA with PEG-based block catiomers had dense PEG palisades, revealing no formation of aggregates without visible interaction with platelets during circulation. This is the first report of in situ monitoring and quantification of the availability of PEGylation to prevent polyplexes from agglomeration over time in the blood circulation. This shows the high utility of IVRTCLSM in drug and gene delivery research.

We describe the preparation of well-defined multicompartment micelles from polybutadiene-block-poly(1-methyl-2-vinyl pyridinium methyl sulfate)-block-poly(methacrylic acid) (BVqMAA) triblock terpolymers and their use as advanced drug delivery systems for photodynamic therapy (PDT). A porphyrazine derivative was incorporated into the hydrophobic core during self-assembly and served as a model drug and fluorescent probe at the same time. The initial micellar corona is formed by negatively charged PMAA and could be gradually changed to poly(ethylene glycol) (PEG) in a controlled fashion through interpolyelectrolyte complex formation of PMAA with positively charged poly(ethylene glycol)-block-poly(L-lysine) (PLL-b-PEG) diblock copolymers. At high degrees of PEGylation, a compartmentalized micellar corona was observed, with a stable bottlebrush-on-sphere morphology as demonstrated by cryo-TEM measurements. By in vitro cellular experiments, we confirmed that the porphyrazine-loaded micelles were PDT-active against A549 cells. The corona composition strongly influenced their in vitro PDT activity, which decreased with increasing PEGylation, correlating with the cellular uptake of the micelles. Also, a PEGylation-dependent influence on the in vivo blood circulation and tumor accumulation was found. Fully PEGylated micelles were detected for up to 24 h in the bloodstream and accumulated in solid subcutaneous A549 tumors, while non- or only partially PEGylated micelles were rapidly cleared and did not accumulate in tumor tissue. Efficient tumor growth suppression was shown for fully PEGylated micelles up to 20 days, demonstrating PDT efficacy in vivo.

Adequate retention in systemic circulation is the preliminary requirement for systemic gene delivery to afford high bioavailability into the targeted site. Polyplex micelle formulated through self-assembly of oppositely-charged poly(ethylene glycol) (PEG)-polycation block copolymer and plasmid DNA has gained tempting perspective upon its advantageous core-shell architecture, where outer hydrophilic PEG shell offers superior stealth behaviors. Aiming to promote these potential characters toward systemic applications, we strategically introduced hydrophobic cholesteryl moiety at the ω-terminus of block copolymer, anticipating to promote not only the stability of polyplex structure but also the tethered PEG crowdedness. Moreover, Mw of PEG in the PEGylated polyplex micelle was elongated up to 20 kDa for expecting further enhancement in PEG crowdedness. Furthermore, cyclic RGD peptide as ligand molecule to integrin receptors was installed at the distal end of PEG in order for facilitating targeted delivery to the tumor site as well as promoting cellular uptake and intracellular trafficking behaviors. Thus constructed cRGD conjugated polyplex micelle with the elevated PEG shielding was challenged to a modeled intractable pancreatic cancer in mice, achieving potent tumor growth suppression by efficient gene expression of antiangiogenic protein (sFlt-1) at the tumor site.

Small interfering RNA (siRNA) therapies administered by intravenous injection have great potential for cancer treatments. E6 and E7 oncogenes of human papillpmavirus (HPV) are known to cause human cervical cancer, because E6 degrades tumor suppressor proteins such as p53, and E7 inactivates the performance of pRB tumor suppressor protein. Thus E6 and E7 oncogenes of HPV are supposed to be good targets of gene therapy against cervical cancers. However, therapeutic siRNA requires a delivery system for transport from the bloodstream into the cytoplasm of cancer cells to perform the function of gene silencing because siRNA is a large water-soluble polyion that can't easily diffuse across cell membrane. Furthermore, enzymatic activity in blood stream causes degradation of siRNA. Macromolecular carriers are supposed to improve the performance of drugs by prolonged blood circulation and preferential tumor accumulation due to the enhanced permeability and retention (EPR) effect. We applied nanosized polymeric micelles that deliver siRNA to solid tumors and elicit a therapeutic effect. Stable multifunctional micelle structures of 45 nm in size formed by spontaneous self-assembly of poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL) block copolymers and the cyclo-Arg-Gly-Asp (cRGD) peptide on the PEG terminus were used to incorporate with HPV E6/E7 siRNAs. To evaluate the in vivo antitumor effect of E6/E7 loaded siRNA micelles (E6siRNA/m), subcutaneous xenograft models were established by transplanting human cervical cells (Hela and CaSki) into nude mice. In vivo toxicity of E6siRNA/m was estimated by weight of mice. The accumulation of E6siRNA/m into tumor and circulation of micelles in blood flow were evaluated by intravital real-time confocal laser scanning microscopy (IVRTCLSM). E6siRNA/m injected intravenously into mice with cervical cancer xenografts demonstrated gene silencing in the tumor mass and showed significantly stronger inhibition of tumor growth compared to control siRNA-loaded micelles (control). Also there was no significant difference in the weight of mice between control and E6siRNA/m groups. IVRTCLSM revealed that E6siRNA/m had prolonged blood circulation and enhanced accumulation into tumors. This new technology of gene delivery using micellar nanoparticle could potentially expand the utility of siRNA-based systemic gene therapies for cervical cancer treatments. Citation Format: Haruka Nishida, Matsumoto Yoko, Christie R. James, Kawana Kei, Inoue Tomoko, Taguchi Ayumi, Nagasaka Kazunori, Oda Katsutoshi, Miyata Kanjiro, Matsumoto Yu, Nishiyama Nobuhiro, Kataoka Kazunori, Fujii Tomoyuki. Analysis of antitumor effect of human papillomavirus E6 siRNA-loaded polymeric micelles on human cervical cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4510. doi:10.1158/1538-7445.AM2013-4510

We describe the development and application of intravital confocal micro-videography to visualize entrance, distribution, and clearance of drugs within various tissues and organs. We use a Nikon A1R confocal laser scanning microscope system attached to an upright ECLIPSE FN1. The Nikon A1R allows simultaneous four channel acquisition and speed of 30 frames per second while maintaining high resolution of 512 × 512 scanned points. The key techniques of our intravital imaging are (1) to present a flat and perpendicular surface to the objective lens, and (2) to expose the subject with little or no bleeding to facilitate optical access to multiple tissues and organs, and (3) to isolate the subject from the body movement without compressing the blood vessels, and (4) to insert a tail vein catheter for timed injection without moving the subject. Ear lobe dermis tissue was accessible without surgery. Liver, kidney, and subcutaneous tumor were accessed following exteriorization through skin incision. In order to image initial extravasations of compounds into tissue following intravenous injection, movie acquisition was initialized prior to drug administration. Our technique can serve as a powerful tool for investigating biological mechanisms and functions of intravenously injected drugs, with both spatial and temporal resolution.